Katja Blanke

Cyclic Mechanical Stretch Induces Cardiomyocyte Orientation and Polarization of the Gap Junction Protein Connexin43

Rationale: Cyclic mechanical stretch (CMS) is an important physiological and pathological factor in the heart. Objective: We examined whether CMS can affect localization of gap junctions with regard to the cell axis. Methods and Results: Neonatal rat cardiomyocytes were cultured (7 days) on flexible 6-well plates. Thereafter, cells were kept static or stimulated with CMS (1 Hz; 0, 10, 20% elongation) for 0, 24, or 48 hours (with or without 10 &mgr;mol/L PD98059, 5 &mgr;mol/L BIM I (bisindolylmaleimide I), 2 &mgr;mol/L H8 [N-(2-methlyamino-ethyl)-5-isoquinoline-sulfonamid], or 0.1 &mgr;mol/L angiotensin II. Additionally, cells were exposed to 24 hours of CMS followed by 24 hours of static recovery. CMS (24 hour, 10%) induced elongation of the cardiomyocytes and orientation 79±8° toward the stretch direction. Moreover, the distribution of connexin (Cx)43 together with N-cadherin changed, so that both proteins were accentuated at the cell poles, whereas in nonstretched cells, they were distributed around the cell without preferential localization. Additional angiotensin II reduced polar Cx43 accentuation. The CMS-induced changes in Cx43 were reversible within 24 hours after end of stretch, and could be completely prevented by the MEK1/2 inhibitor PD98059 but not by BIM I or H8. Moreover, stretch resulted in Cx43 protein and Cx43-mRNA upregulation and in a significant upregulation of the phosphorylated forms of ERK1/2, glycogen synthase kinase 3&bgr; and AKT. Furthermore, CMS resulted in a significant increase of the transcription factors activator protein 1 and CREB (cAMP response element–binding protein) in the nucleus. Conclusions: CMS results in self-organization of cardiomyocytes leading to elongated cells orientated transverse to the stretch axis, enhanced Cx43 expression and Cx43 accentuation at the cell poles. The Cx43-changes seem to depend on the ERK1/2 signaling cascade.

Right or Left Ventricular Pacing in Young Minipigs With Chronic Atrioventricular Block Long-Term In Vivo Cardiac Performance, Morphology, Electrophysiology, and Cellular Biology

Background— Left ventricular (LV) dyssynchrony may occur as a result of right ventricular (RV) pacing and is a known risk factor for the development of heart failure. In children with complete atrioventricular block, pacing-induced dyssynchrony lasting for decades might be especially deleterious for LV function. To determine the hemodynamic and ultrastructural remodeling after either RV free wall or LV apical pacing, we used a chronic minipig model. Methods and Results— Fourteen piglets 8 weeks of age underwent atrioventricular node ablation and were paced from either the RV free wall or the LV apex at 120 bpm for 1 year (7 age-matched minipigs served as controls with spontaneous heart rates of 104±5 bpm). Echocardiographic examinations, pressure-volume loops, patch-clamp investigations, and examinations of connexin43, calcium-handling proteins, and histomorphology were carried out. RV free wall–paced minipigs exhibited significantly more LV dyssynchrony than LV apex–paced animals, which was accompanied by worsening of LV function (maximum LV mechanical delay/LV ejection fraction: RV free wall pacing, 154±36 ms/28±3%, LV apical pacing, 52±19 ms/45±2%, control 47±14 ms/62±1%; P=0.0001). At the cellular level, both pacemaker groups exhibited a significant reduction in L-type calcium and peak sodium current, shortening of action potential duration and amplitude, increased cell capacity, and alterations in the calcium-handling proteins that were similar for RV free wall– and LV apex–paced animals. Conclusions— The observed molecular remodeling seemed to be more dependent on heart rate than on dyssynchrony. LV apical pacing is associated with less dyssynchrony, a more physiological LV contraction pattern, and preserved LV function as opposed to RV free wall pacing.Background— Left ventricular (LV) dyssynchrony may occur as a result of right ventricular (RV) pacing and is a known risk factor for the development of heart failure. In children with complete atrioventricular block, pacing-induced dyssynchrony lasting for decades might be especially deleterious for LV function. To determine the hemodynamic and ultrastructural remodeling after either RV free wall or LV apical pacing, we used a chronic minipig model. Methods and Results— Fourteen piglets 8 weeks of age underwent atrioventricular node ablation and were paced from either the RV free wall or the LV apex at 120 bpm for 1 year (7 age-matched minipigs served as controls with spontaneous heart rates of 104±5 bpm). Echocardiographic examinations, pressure-volume loops, patch-clamp investigations, and examinations of connexin43, calcium-handling proteins, and histomorphology were carried out. RV free wall–paced minipigs exhibited significantly more LV dyssynchrony than LV apex–paced animals, which was accompanied by worsening of LV function (maximum LV mechanical delay/LV ejection fraction: RV free wall pacing, 154±36 ms/28±3%, LV apical pacing, 52±19 ms/45±2%, control 47±14 ms/62±1%; P =0.0001). At the cellular level, both pacemaker groups exhibited a significant reduction in L-type calcium and peak sodium current, shortening of action potential duration and amplitude, increased cell capacity, and alterations in the calcium-handling proteins that were similar for RV free wall– and LV apex–paced animals. Conclusions— The observed molecular remodeling seemed to be more dependent on heart rate than on dyssynchrony. LV apical pacing is associated with less dyssynchrony, a more physiological LV contraction pattern, and preserved LV function as opposed to RV free wall pacing. # Clinical Perspective {#article-title-39}

Role of connexins in human congenital heart disease: the chicken and egg problem

Inborn cardiac diseases are among the most frequent congenital anomalies and are the main cause of death in infants within the first year of age in industrialized countries when not adequately treated. They can be divided into simple and complex cardiac malformations. The former ones, for instance atrial and ventricular septal defects, valvular or subvalvular stenosis or insufficiency account for up to 80% of cardiac abnormalities. The latter ones, for example transposition of the great vessels, Tetralogy of Fallot or Shone’s anomaly often do not involve only the heart, but also the great vessels and although occurring less frequently, these severe cardiac malformations will become symptomatic within the first months of age and have a high risk of mortality if the patients remain untreated. In the last decade, there is increasing evidence that cardiac gap junction proteins, the connexins (Cx), might have an impact on cardiac anomalies. In the heart, mainly three of them (Cx40, Cx43, and Cx45) are differentially expressed with regard to temporal organogenesis and to their spatial distribution in the heart. These proteins, forming gap junction channels, are most important for a normal electrical conduction and coordinated synchronous heart muscle contraction and also for the normal embryonic development of the heart. Animal and also some human studies revealed that at least in some cardiac malformations alterations in certain gap junction proteins are present but until today no particular gap junction mutation could be assigned to a specific cardiac anomaly. As gap junctions have often been supposed to transmit growth and differentiation signals from cell to cell it is reasonable to assume that they are somehow involved in misdirected growth present in many inborn heart diseases playing a primary or contributory role. This review addresses the potentional role of gap junctions in the development of inborn heart anomalies like the conotruncal heart defects.

Cardiac gap junction channels are upregulated by metoprolol: an unexpected effect of beta-blockers.

Background/Aims: Since β-adrenoceptors have been shown to affect cardiac gap junction channels, we wanted to elucidate the possible effect of metoprolol on the gap junction protein connexin-43, using racemic RS-metoprolol or the isomer R-metoprolol (no β-adrenoceptor blockade) or S-metoprolol (β1-adrenoceptor blocker). Methods: Cultured neonatal rat cardiomyocytes were exposed to either RS-metoprolol or R-metoprolol or S-metoprolol (0.1 µmol/l each) without or with additional isoprenaline (0.1 µmol/l) treatment for 24 h. Results: The β-blocker treatment did not alter the frequency of spontaneously beating cardiomyocytes, whereas sole isoprenaline administration significantly enhanced the beating frequency by about 40%. This rise could be blocked by concomitant treatment with S- or RS-metoprolol but not with R-metoprolol. Connexin-43 protein was significantly enhanced by isoprenaline and by R-, S- or RS-metoprolol treatment alone as well as with the combined administration of isoprenaline and R-, S- or RS-metoprolol. Phospho-ERK1 and connexin-43 mRNA were significantly increased by isoprenaline application alone, whereas R-, S- or RS-metoprolol alone or in combination with isoprenaline exhibited no effect. Conclusion: Both isomers of metoprolol upregulate connexin-43 in cultured cardiomyocytes by a β-adrenoceptor-independent mechanism. Since the enhanced presence of connexin-43 in cell membranes under metoprolol was not accompanied by enhanced connexin-43 mRNA, we assume that the metoprolol effect involves reduced connexin-43 degradation.

Mind the gap! Connexins and pannexins in physiology, pharmacology and disease

Among other aspects it is the communication which makes the difference between a crowd of individuals and a society. Similarly, a key feature of an organism or of organs is the communication between their individual cells realized by mediators, hormones, and by direct intercellular communication via gap junction channels allowing the transmission of electrical signals and the exchange of small molecules to regulate growth and differentiation. This enables the organ or the organism to adapt very efficiently to the actual needs. Due to the important role of gap junction intercellular communication (GJIC) for the correct functioning of organs, and tissues a tight regulation of the expression of gap junction channel proteins, the connexins, their localization, and function is required. Besides connexins, another group of proteins, the pannexins, showing many molecular similarities with connexins have been identified. They seem to form hemichannels which may regulate cytosolic homeostasis or the release of small molecules. The present issue provides a comprehensive picture of recent developments and current research in this fascinating, fast developing area comprising review and original research articles on both connexins and pannexins written by leading experts in their research areas. The articles are organized in three parts:

Role of connexins in infantile hemangiomas

The circulatory system is one of the first systems that develops during embryogenesis. Angiogenesis describes the formation of blood vessels as a part of the circulatory system and is essential for organ growth in embryogenesis as well as repair in adulthood. A dysregulation of vessel growth contributes to the pathogenesis of many disorders. Thus, an imbalance between pro- and antiangiogenic factors could be observed in infantile hemangioma (IH). IH is the most common benign tumor during infancy, which appears during the first month of life. These vascular tumors are characterized by rapid proliferation and subsequently slower involution. Most IHs regress spontaneously, but in some cases they cause disfigurement and systemic complications, which requires immediate treatment. Recently, a therapeutic effect of propranolol on IH has been demonstrated. Hence, this non-selective β-blocker became the first-line therapy for IH. Over the last years, our understanding of the underlying mechanisms of IH has been improved and possible mechanisms of action of propranolol in IH have postulated. Previous studies revealed that gap junction proteins, the connexins (Cx), might also play a role in the pathogenesis of IH. Therefore, affecting gap junctional intercellular communication is suggested as a novel therapeutic target of propranolol in IH. In this review we summarize the current knowledge of the molecular processes, leading to IH and provide new insights of how Cxs might be involved in the development of these vascular tumors.

Effect of Angiotensin(1-7) on Heart Function in an Experimental Rat Model of Obesity

Aim: Obesity is a risk factor for the development of cardiovascular diseases. Recently it was shown that overexpression of the Mas-receptor antagonist angiotensin(1-7) could prevent from diet-induced obesity. However, it remained unclear whether diet-induced obesity and angiotensin(1-7) overexpression might also have effects on the cardiovascular system in these rats. Methods:Twenty three male Sprague Dawley rats were fed with standard chow (SD+chow, n = 5) or a cafeteria diet (SD+CD, n = 6) for 5 months. To investigate the effect of angiotensin(1-7) transgenic rats, expressing an angiotensin(1-7)-producing fusion protein in testis were used. These transgenic rats also received a 5 months feeding period with either chow (TGR+chow, n = 6) or cafeteria diet (TGR+CD, n = 6), respectively. Hemodynamic measurements (pressure-volume loops) were carried out to assess cardiac function and blood pressure. Subsequently, hearts were explanted and investigated according to the Langendorff technique. Furthermore, cardiac remodeling in these animals was investigated histologically. Results:After 5 months cafeteria diet feeding rats showed a significantly increased body weight, which could be prevented in transgenic rats. However, there was no effect on cardiac performance after cafeteria diet in non-transgenic and transgenic rats. Moreover, overexpression of angiotensin(1-7) deteriorated cardiac contractility as indicated by impaired dp/dt. Furthermore, histological analysis revealed that cafeteria diet led to myocardial fibrosis in both, control and transgenic rats and this was not inhibited by an overproduction of angiotensin(1-7). Conclusion:These results indicate that an overexpression of circulating angiotensin(1-7) prevents a cafeteria diet-induced increase in body weight, but does not affect cardiac performance in this experimental rat model of obesity. Furthermore, overexpression of angiotensin(1-7) alone resulted in an impairment of cardiac function.

Right or Left Ventricular Pacing in Young Minipigs With Chronic Atrioventricular BlockClinical Perspective

Background— Left ventricular (LV) dyssynchrony may occur as a result of right ventricular (RV) pacing and is a known risk factor for the development of heart failure. In children with complete atrioventricular block, pacing-induced dyssynchrony lasting for decades might be especially deleterious for LV function. To determine the hemodynamic and ultrastructural remodeling after either RV free wall or LV apical pacing, we used a chronic minipig model. Methods and Results— Fourteen piglets 8 weeks of age underwent atrioventricular node ablation and were paced from either the RV free wall or the LV apex at 120 bpm for 1 year (7 age-matched minipigs served as controls with spontaneous heart rates of 104±5 bpm). Echocardiographic examinations, pressure-volume loops, patch-clamp investigations, and examinations of connexin43, calcium-handling proteins, and histomorphology were carried out. RV free wall–paced minipigs exhibited significantly more LV dyssynchrony than LV apex–paced animals, which was accompanied by worsening of LV function (maximum LV mechanical delay/LV ejection fraction: RV free wall pacing, 154±36 ms/28±3%, LV apical pacing, 52±19 ms/45±2%, control 47±14 ms/62±1%; P=0.0001). At the cellular level, both pacemaker groups exhibited a significant reduction in L-type calcium and peak sodium current, shortening of action potential duration and amplitude, increased cell capacity, and alterations in the calcium-handling proteins that were similar for RV free wall– and LV apex–paced animals. Conclusions— The observed molecular remodeling seemed to be more dependent on heart rate than on dyssynchrony. LV apical pacing is associated with less dyssynchrony, a more physiological LV contraction pattern, and preserved LV function as opposed to RV free wall pacing.Background— Left ventricular (LV) dyssynchrony may occur as a result of right ventricular (RV) pacing and is a known risk factor for the development of heart failure. In children with complete atrioventricular block, pacing-induced dyssynchrony lasting for decades might be especially deleterious for LV function. To determine the hemodynamic and ultrastructural remodeling after either RV free wall or LV apical pacing, we used a chronic minipig model. Methods and Results— Fourteen piglets 8 weeks of age underwent atrioventricular node ablation and were paced from either the RV free wall or the LV apex at 120 bpm for 1 year (7 age-matched minipigs served as controls with spontaneous heart rates of 104±5 bpm). Echocardiographic examinations, pressure-volume loops, patch-clamp investigations, and examinations of connexin43, calcium-handling proteins, and histomorphology were carried out. RV free wall–paced minipigs exhibited significantly more LV dyssynchrony than LV apex–paced animals, which was accompanied by worsening of LV function (maximum LV mechanical delay/LV ejection fraction: RV free wall pacing, 154±36 ms/28±3%, LV apical pacing, 52±19 ms/45±2%, control 47±14 ms/62±1%; P =0.0001). At the cellular level, both pacemaker groups exhibited a significant reduction in L-type calcium and peak sodium current, shortening of action potential duration and amplitude, increased cell capacity, and alterations in the calcium-handling proteins that were similar for RV free wall– and LV apex–paced animals. Conclusions— The observed molecular remodeling seemed to be more dependent on heart rate than on dyssynchrony. LV apical pacing is associated with less dyssynchrony, a more physiological LV contraction pattern, and preserved LV function as opposed to RV free wall pacing. # Clinical Perspective {#article-title-39}

Right or Left Ventricular Pacing in Young Minipigs With Chronic Atrioventricular BlockClinical Perspective: Long-Term In Vivo Cardiac Performance, Morphology, Electrophysiology, and Cellular Biology

Background— Left ventricular (LV) dyssynchrony may occur as a result of right ventricular (RV) pacing and is a known risk factor for the development of heart failure. In children with complete atrioventricular block, pacing-induced dyssynchrony lasting for decades might be especially deleterious for LV function. To determine the hemodynamic and ultrastructural remodeling after either RV free wall or LV apical pacing, we used a chronic minipig model. Methods and Results— Fourteen piglets 8 weeks of age underwent atrioventricular node ablation and were paced from either the RV free wall or the LV apex at 120 bpm for 1 year (7 age-matched minipigs served as controls with spontaneous heart rates of 104±5 bpm). Echocardiographic examinations, pressure-volume loops, patch-clamp investigations, and examinations of connexin43, calcium-handling proteins, and histomorphology were carried out. RV free wall–paced minipigs exhibited significantly more LV dyssynchrony than LV apex–paced animals, which was accompanied by worsening of LV function (maximum LV mechanical delay/LV ejection fraction: RV free wall pacing, 154±36 ms/28±3%, LV apical pacing, 52±19 ms/45±2%, control 47±14 ms/62±1%; P=0.0001). At the cellular level, both pacemaker groups exhibited a significant reduction in L-type calcium and peak sodium current, shortening of action potential duration and amplitude, increased cell capacity, and alterations in the calcium-handling proteins that were similar for RV free wall– and LV apex–paced animals. Conclusions— The observed molecular remodeling seemed to be more dependent on heart rate than on dyssynchrony. LV apical pacing is associated with less dyssynchrony, a more physiological LV contraction pattern, and preserved LV function as opposed to RV free wall pacing.Background— Left ventricular (LV) dyssynchrony may occur as a result of right ventricular (RV) pacing and is a known risk factor for the development of heart failure. In children with complete atrioventricular block, pacing-induced dyssynchrony lasting for decades might be especially deleterious for LV function. To determine the hemodynamic and ultrastructural remodeling after either RV free wall or LV apical pacing, we used a chronic minipig model. Methods and Results— Fourteen piglets 8 weeks of age underwent atrioventricular node ablation and were paced from either the RV free wall or the LV apex at 120 bpm for 1 year (7 age-matched minipigs served as controls with spontaneous heart rates of 104±5 bpm). Echocardiographic examinations, pressure-volume loops, patch-clamp investigations, and examinations of connexin43, calcium-handling proteins, and histomorphology were carried out. RV free wall–paced minipigs exhibited significantly more LV dyssynchrony than LV apex–paced animals, which was accompanied by worsening of LV function (maximum LV mechanical delay/LV ejection fraction: RV free wall pacing, 154±36 ms/28±3%, LV apical pacing, 52±19 ms/45±2%, control 47±14 ms/62±1%; P =0.0001). At the cellular level, both pacemaker groups exhibited a significant reduction in L-type calcium and peak sodium current, shortening of action potential duration and amplitude, increased cell capacity, and alterations in the calcium-handling proteins that were similar for RV free wall– and LV apex–paced animals. Conclusions— The observed molecular remodeling seemed to be more dependent on heart rate than on dyssynchrony. LV apical pacing is associated with less dyssynchrony, a more physiological LV contraction pattern, and preserved LV function as opposed to RV free wall pacing. # Clinical Perspective {#article-title-39}